Extracting, Gene Cloning And Expression And Functional Analysis Of Spore Surface Proteins From Fusarium Verticillioide

Parasitism is a parasitic way between parasitic fungi and host fungi, it is an ubiquitous phenomenon between the fungus in the nature. Fusarium verticillioide is one of the important plant pathogenic fungus and host fungus, while Olpitrichum tenellum is one of the parasitic fungus on the F. verticillioide which can recognizes、interactes and then lives on the bodies of the F. verticillioide. It was proved that the protein of spore suface had an important role in the process of recognization between the parasitic fungi and host fungi, so, there is an important scientific significance to research it.Hydrophobins are a kind of secretory and small molecule protein, which exist in the surface of hyphae and spore structure of filamentous fungi. Hydrophobins contain eight homocysteine remnants and a signal peptide. They are a class of amphiphilic proteins and play an extremely important biological function in growth of fungi.This paper first used different methods (TCA method, SDS boilling method, sucrose permeability method and freeze-thaw method) to extract proteins of spore surface from F. verticillioide. SDS-PAGE electrophoresis showed that we obtained seven bands by the method of TCA. Molecular mass of one of the seven bands was about12kDa. Four proteins were been extracted by boiling with SDS. Molecular mass of two bands was about12kDa. We extracted fourteen and six bands by sucrose permeability method and freeze-thaw method respectively. The former had25kDa band and other bands whose molecular weight were above the50kDa, while the latter didn’t have. Although we obtained different proteins by the four methods, but there were the same bands by SDS-PAGE. TCA and SDS boilling method mainly extracted small molecular weight proteins, and sucrose permeability and freeze-thaw method can extract other proteins in addition to the small molecular weight proteins.To obtain hydrophobin genes from F. verticillioide, degenerate primers were designed on the sequence of GenBank, and two cDNA fragments were obtained through RT-PCR. The full length of HydⅠ and HydⅡ cDNA gene were381bp and372bp, which contained an ORF of127and124amino acids. The genes of HydⅠ and HydⅡ from F. verticillioide and expression vector pPIC9K were digested ligated in vitro to construct eukaryocyte expression plasmid pPIC9K/HydI and pPIC9K/HydII. The recombinant vectors, pPIC9K/HydI and pPIC9K/HydII were transformed to Pichia pastor is GS115competent cell. The recombinant P. pastoris G-HydⅠ and G-HydII were obtained and the HydⅠ and HydⅡ expressed were purified. The molecular weight of hydrophobinl is11.7kDa, and isoelectric point is6.08theoretically. The molecular weight of hydrophobinⅡ is11.2kDa, and isoelectric point is7.61theoretically. After purified proteins interacted with spores of F. verticillioide and O. tenellum, we found that the two proteins can aggregate the spores of F. verticillioide and O. tenellum. The optimal pH of HydⅠ to aggregate the spores was6.0, while HydⅡ was7.0, the optimal time was15min, while HydⅡ was10min.After the study of hydrophobin agglomeration with the spores of F. verticillioide and O. tenellum, we selected other spores of Trichoderma viride、Thermomyces lanuginosu and Fusarium kyushuense for further agglomeration studies. The results showed that the hydrophobins can aggregate the spores of T. lanuginosus and F. kyushuense, while the hydrophobins can inhibit agglomeration T.viride spores.To knock out the hydrophobin gene HydⅡ from F. verticillioide, the expression vector pROKII was ligated to hygromycin B resistance gene cassette (PtrpC-hph-TtrpC) with the fungus promoter from plasmid pUCATPH, and constructed The vector pROKII-S-PtrpC-hph-TtrpC-X.